There was an overall significant increase in IL-1β from pre- to post-swimming , but no significant interaction. IL-10 significantly increased in both groups . IL-6 and TNF-α increased significantly  in adults, but not in adolescents . Adults showed a small, but significant decrease in irisin (−5%), with no change in adolescents. The lack of an IL-6, TNF-α and irisin response to intense swimming in adolescent swimmers may suggest a blunted inflammatory and myokine response following high intensity exercise in trained youth.Swimming is a popular youth sport that is considered beneficial for cardiovascular fitness. However, the potential inflammatory outcomes of high intensity swimming in younger swimmers are unclear, as is the response of irisin, a myokine released during exercise with anti-inflammatory properties. This study compared the plasma concentrations of interleukins 1-beta (IL-1β), 6 (IL-6), 10 (IL-10), tumor necrosis factor alpha (TNF-α) and irisin in response to intense swimming between adolescent and adult male swimmers. Thirty-two swimmers  completed a high intensity interval swimming trial. At rest, only TNF-α was higher (33%,  Intense exercise can significantly influence several inflammatory markers, while the magnitude and direction of changes in specific cytokines, may be mediated by the mode and intensity of the activity, as well as the age and training status of participants ,2. For eInterleukin 1-beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor alpha (TNF-α) are known pro-inflammatory cytokines that are released in the early stages of the inflammation process in response to muscle injury to initiate the muscle regeneration process . IL-10 iMost available data on the inflammatory response to exercise relate to prolonged, moderate exercise. However, the increase in cytokine levels following repeated bouts of intense exercise, may be attenuated compared with the response following the initial bout . It is pParticipants in the current study included 16 adolescent (11–15 years old) and 16 adult (18–30 years old) male swimmers, who trained and competed for at least four years, were free of injuries and any medical condition that prevented them from participating and were not taking medications or nutritional supplements. Swimmers were recruited from two varsity teams and five local swim clubs in Southern Ontario as part of a larger study designed to examine the effect of post-exercise whey protein consumption on subsequent exercise performance, muscle damage, and inflammation . Data prAll participants and their parents/guardians were provided a thorough explanation of the study’s purpose, procedures, and potential risks. Consent was obtained from all participants and their respective parent/guardian where applicable. The study procedures and secondary analysis presented herein were cleared by the Research Ethics Boards of Brock University  and the Canadian Sports Institute of Ontario (REB# 2017-01).The study employed a parallel cross-sectional design which included two visits. The first visit was an information session, which also included anthropometric measurements. Details of the measurements can be found in McKinlay et al. . In brieBetween visits 1 and 2, participants were instructed to refrain from any exercise for a minimum of 48 h prior to visit 2. The second visit included the swimming trial and two blood samples: one fasted , a resting  sample collected at 06:00 h, and one post-swimming sample collected within 15 min following swimming. Both the swimming trial and the blood draws are described below. Immediately after the resting blood sample and ~1 h before swimming, participants were provided a standardized breakfast of ~300–500 kcal (depending on body mass), which included a granola bar, muffin, fruit (banana or apple) and a juice box or water. Details of the ingredients of the breakfast can be found in McKinlay et al. .The swimming trial commenced ~1.5 h after the pre-exercise blood draw  and it was designed for the purposes of the larger study to mirror a day of swimming competition. Details of the larger study’s protocol and measurements can be found in McKinlay et al. . Brieflyg at 4 °C using a benchtop centrifuge for 10 min. Serum and plasma were then aliquoted into pre-labeled Eppendorf tubes to be stored at −80 °C until analysis.In order to control for circadian rhythm, all testing was performed in the morning hours. Blood samples were collected at rest , and immediately (within 15 min) following the swimming trial (09:00–10:00 h). Venous samples (10 mL) were drawn from the antecubital fossa using a standard venipuncture technique. All samples were centrifuged at 1405× Plasma concentrations of IL-1β, IL-6, IL-10 and TNF-α were measured in duplicates using multiplex magnetic bead kits . The average in-house inter- and intra-assay coefficients of variation (CV) for IL-6, IL-10, IL-1β and TNF-α were 8.1% and 4.3%, 6.5% and 4.3%, 4.6% and 7.3%, 8% and 5.3%, respectively. Irisin was also measured in duplicate using an irisin recombinant assay kit . The average in-house inter- and intra-assay CV for irisin were 1.2% and 4%, respectively.PV) were calculated using the formula by Van Beaumont [Hct1 is hematocrit at baseline, and Hct2 is hematocrit at each post-baseline measurement. The %ΔPV was then used to adjust the post-swimming plasma concentrations of inflammatory cytokines  and irisin using the formula: 100+%ΔPV/100.For every blood draw, hematocrit was measured in triplicates by the same investigator using microhematocrit capillary tubes treated with heparin . Relative plasma volume changes .All variables were first checked for normality using the Kolmogorov-Smirnov test; variables were also checked for skewness and kurtosis of ±3. In addition, visual screening of histograms for symmetry was performed. IL-1β, IL-6, IL-10 and irisin concentrations were not normally distributed, and were log transformed for further analysis. Group differences in physical characteristics, resting cytokines and irisin levels were analyzed using independent t-tests. A two-way (group-by-time) ANOVA for repeated measures was used to examine changes in cytokines  and irisin levels (pre- to post-swimming). In the event of a significant interaction, post-hoc comparisons were performed within each group using paired t-tests. The baseline characteristics are reported as mean ± standard deviation (SD) and cytokine concentrations at rest are reported as mean ± standard error (SE) in descriptive tables. The results of the ANOVA repeated measurements are shown reporting F, p = 0.02) in the adolescent male swimmers compared to the adults , reflecting an overall large increase from pre to post-exercise, with no significant group effect  and no significant group-by-time interaction . Thus, although IL-1β increased by 3% in adolescents versus 24% in adults, this difference was not statistically significant , but a significant time effect  and a large, significant group-by-time interaction , reflecting a significant increase following intense swimming in the adult swimmers , which was not the case in adolescents , with no significant group effect  or group-by-time interaction . For TNF-α, we found no effect for group , a significant time effect , and a significant group-by-time interaction , which reflects a significant increase in the adults  with a non-significant decrease  in the adolescents , but not for the group , and a significant interaction  were also found for irisin, reflecting a small yet significant decrease in adults , with no change in the adolescents (nificant . IL-6 shlescents . There wlescents . A signilescents .2 ranging from 0.14 to 0.35). Specifically, IL-1β, IL-6, IL-10 and TNF-α significantly increased while irisin decreased from pre- to post-swimming in the adults, but in the adolescents, only the anti-inflammatory IL-10 increased significantly in response to high intensity swimming.To our knowledge, this is the first study to directly compare the response of anti- and pro-inflammatory cytokines, and irisin to high intensity interval swimming between two age groups of competitive male swimmers. Previous studies typically examined age-related differences in cytokine and irisin responses to acute exercise in non-athletic populations, who on one hand are accustomed to the specific exercise, but on the other hand are exposed to chronic high intensity, non-eccentric, exercise with little recovery time. Overall, we observed significant exercise-induced increases in all cytokines, and a decrease in irisin, in adults, but these changes were blunted or not evident in the adolescent swimmers, as evidenced by large interaction effects , which is of lower intensity and involves fewer muscle groups than impacted with our swimming protocol. Regardless of these differences, and the fact that they also supplemented participants with a 6% carbohydrate beverage or placebo, both studies concluded that the inflammatory response to exercise in children and adolescents is lower than what is observed in adults, regardless of training status. According to these authors, children’s IL-6 response to sustained, vigorous exercise tends to be approximately 50% lower than in adults, suggesting that young boys may exhibit a blunted inflammatory response following vigorous exercise [In the adolescent swimmers, our post-swimming results agree with Timmons et al.  in that exercise . This bl2max [The blunted inflammatory response to vigorous exercise in children could also be a protective mechanism during growth. However, we did not measure IGF-1 or other circulating growth factors to support this speculation. A study by Nemet et al.  reported2max . However2max ,34. In a2max . It is pIn terms of irisin, we observed a small, but significant, decrease in adults with no significant changes in adolescents following the intense swimming trial. These results are difficult to explain. One explanation could be related to the mode of the exercise being performed. Two previous studies in children and adolescents reported increases in irisin immediately following running and cycling ,21 whileThe main strength of this study is the consideration of post-exercise plasma volume changes that adds rigour to the experimental design. The interpretation of any post-exercise result is conditional upon establishing whether exercise is effective in eliciting a response beyond the effects of hemoconcentration. A limitation to this current study is its small sample size, which, although similar to that of previous studies on the same topic, may have limited the power to detect significant changes. Another limitation of the study is that the findings are only applicable to the mode of exercise used, i.e., swimming, and may not be applicable to other forms of land-based overground activity such as running. Indeed, it is possible that a land-based activity that is predominantly eccentric and high impact, and as such applies an overall higher stress to the musculoskeletal system, may result to a greater cytokine response across ages. Future studies should consider comparing swimming with equivalent overground land-based running in both male and female athletes for a more complete picture of the inflammatory response to intense exercise.In summary, a high intensity interval swimming trial did not induce significant acute increases in inflammatory cytokines, with the exception of anti-inflammatory IL-10, in adolescent male swimmers, while all cytokines, including IL-1β, IL-6, IL-10 and TNF-α, significantly increased in adult swimmers following the same swimming trial. Likewise, irisin levels did not significantly change in adolescent swimmers, whereas in adult swimmers the levels significantly decreased following high intensity swimming. Our results demonstrate that adolescent athletes display a blunted inflammatory response in contrast to what usually follows exercise in adults. It is possible that training-induced adaptations in adolescents follow a different timescale compared with adults. These results provide a mechanistic insight as to why young athletes require less time to recover from intense exercise and suggest that a different recovery strategy may be effective in this group.


To find the expression of KISS-1 and G protein-coupled receptor 54 in rats testis from PND 21st to 56th.2max, 1 h/day, 5 days/week) intervention and were randomly divided into group C, CE, HC, or HE. Sample time points were set on the PND 21st, 35th, 43rd, and 56th. The testicular testosterone and the mRNA content, and protein content of KISS-1 and GPR54 in testis tissue were detected by ELISA, RT-qPCR, and Western blotting.128 three-week-old weaned rats underwent high-fat diet and exercise (60–70% VO2max exercise decreased the KISS-1/GPR54 expression level. Exercise intervention improved testicular development in rats with a high-fat diet.(1) The protein of KISS-1 and GPR54 increased gradually during the growing period. KISS-1 mRNA peaked at 35D and GPR54 peaked at 43D. (2) High-fat diet affected the expression of the KISS-1/GPR54 system in rat testis and reduced the expression level of KISS-1 protein. (3) 60–70% VO2max exercise decreased the expression of KISS-1/GPR54, which may be involved in the effects of exercise on high-fat dietary sex hormone disorders.The expression of KISS-1/GPR54 increased during the growing period. High-fat diet can downregulate the protein and gene expression of KISS-1/GPR54 and change the expression trend. 60–70% VO The discovery of the KISS-1/ G protein-coupled receptor 54, GPR 54 system has provided a new way to understand the link between energy and reproduction. In recent years, studies have shown that the axons of KISS-1 neurons attach to GnRH neurons and GPR54 neurons that are located on GnRH neurons. Intervention of KISS-1 or GPR54 gene expression can affect GnRH secretion, which supports the view that the KISS-1/GPR54 system directly regulates GnRH secretion by GnRH neurons and thus regulates gonadal development [Our previous studies found that high-fat diet intervention could upregulate the expression of KISS-1/GPR54 in the hypothalamus of growing male rats, but it could also lead to lipid droplet deposition in testicular tissue and decrease the serum testosterone level.According to its gene expression profile, KISS-1 is mostly highly expressed in the brain, followed by tissues such as placenta, testis, pancreas, liver, and small intestine; GPR54 is also highly expressed in the brain, followed by tissues such as pancreas, placenta, and testis . HoweverRecent data indicates that kisspeptin, encoded by the KISS1 gene, could play a role in transducing metabolic information into the HPG axis, the mechanism that controls reproductive functions , 8. SeveIn recent years, exercise has attracted attention as an effective intervention to reduce energy accumulation, regulate energy imbalance, and reduce body fat to improve the adverse effects of a high-fat diet in various ways. Swimming, moderate-intensity treadmill exercise, wheel running, and other sport forms have been shown to improve rat fatness caused by a high-fat diet by increasing energy consumption, increasing white fat browning, and reducing fat accumulation , 13 For In view of, (1) the KISS-1/GPR54 system being a puberty-initiated control switch; (2) the KISS-1/GPR54 system existing in the gonads, but with an unclear function; (3) Exercise being able to regulate obesity-induced sex hormone disorders, but with an unclear mechanism, we hypothesized that the KISS-1/GPR54 system in gonadal tissue is involved in the regulation of gonadal development during the growing period, and that the KISS-1/GPR54 system is involved in the regulation of sex hormone disorders induced by high-fat diet, and that exercise can regulate these disorders. We studied the changing KISS-1/GPR54 system in testis during the growing period, and then based on this, we explored the possible relationship between KISS-1/GPR54 with testicular tissue testosterone.All experiments were approved by the Department of Zoology Research Committee, Beijing Sport University. 128 three-week-old weaning SD rats were purchased from Beijing Weitonglihua Laboratory Animal Co. Ltd., Beijing (License No. SCXK Beijing 2012-0001). Animals were housed under standard laboratory conditions (light:dark 12 h:12 h) and were provided with pelleted food and water ad libitum.−1; 10% energy from fat, 20% from protein and 70% from carbohydrate; Beijing Huafukang Biotechnology Co. Ltd., LOT No: D12451B); groups HC and HE were fed with high-fat feed with 45 kcal% Fat ,using the same formulation with D12451 from Research Diets, USA.Animals were randomly divided into standard diet group , standard exercise group , high fat diet group  and high fat exercise group . Groups C and CE were fed with control feed, and during the first three days, the durations were 10 min, 20 min, and 30 min. After the habituation period, the incline was raised to 10-degrees for the duration of the training period. Training included warm-up (5 min), training (50 min), and recovery (5 min). The running speeds of warm-up and recovery stages were 10 m/min; the running speed of the training stage was 60–70% vVO2 max, with training 5 days per week. The specific training program was as follows:The rats of each group were classified into following age groups: (a) PND 21st day ; (b) PND 35th day , (c) PND 43rd day , (d) PND 56th day . For the impact of acute exercise, each sample collection was taken at 48 h after the end of the last endurance exercise training. The body mass of each mouse was recorded before killing; 2% sodium pentobarbital was used to anesthetize them by intraperitoneal injection at a dose of 0.25 ml/100 g body weight. After anesthesia, the testes on both sides were taken, the testicular fat and epididymis were removed, and the mass of the two testes was determined. Testis were kept at −80 °C for RT-qPCR and Western blotting.Total RNA was isolated from testes using an RNAprep Pure Tissue Kit  following the manufacturer’s protocol. The time of the on-column DNA digestion was extended from 15 to 45 min to ensure complete removal of genomic DNA. The RNA was reverse transcribed into cDNA using TOYOBO ReverTra Ace qPCR RT Kit  following the protocol provided by the manufacturer. The primer sequences were: KISS1 , GPR54 , 18S . After the two-step PCR reaction procedure, the amplification curve and the melting curve of real-time PCR were confirmed, a standard curve was prepared for PCR quantification, and the gene expression amount of each sample was obtained by CT value.Total protein was extracted from testis using PBS . Protein concentrations were determined using an assay kit . Then, 50 μg of protein lysates were loaded, separated by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane . Membranes were incubated in blocking buffer  for 1 h at 37 °C, followed by hybridization with anti-KISS-1  or anti-GPR54  primary antibody at 4 °C overnight. Then, membranes were hybridized with a horseradish peroxidase-conjugated rabbit immunoglobulin G secondary antibody  for 1 h. Protein bands were detected by chemiluminescence using a Western blotting luminol reagent (SUDGEN). Films were scanned using a Bio-Rad imager and the protein level was semi-quantified using Quantity One 1D image analysis software Bio-Rad 5.2.p < 0.05 as statistically significant and p < 0.01 as statistically significant.The densitometric data are presented as the mean ± SEM. The bands obtained from immunoblots were normalized to GAPDH . All data were tested by a normality test and homogeneity of variance before analysis. After meeting the criteria, the results were statistically analyzed by multivariate analysis of variance. The three-factor analysis of variance was performed by setting the age of the rats , dietary intervention factors , exercise intervention (with or without exercise) as the three factors. If there was a three-factor interaction between the independent variables, a separate effect test was used; if there was no three-factor interaction between the independent variables, then a two-factor interaction analysis was performed, and if there was a two-factor interaction, a separate effect test was performed, taking p < 0.05). The high-fat diet intervention significantly reduced rat GSI, while exercise intervention significantly increased rat GSI, showing that exercise intervention can improve the GSI of high-fat diet rats. The body weight of growing rats as Additional file The results showed that both the testicular mass and GSI increased gradually from early childhood to maturity Figs.  and 2. Ep < 0.05). High-fat diet intervention also downregulated testicular tissue testosterone levels in rats (p < 0.01). Exercise intervention can improve testicular tissue testosterone levels in high-fat diet rats (p < 0.05).Figure p < 0.05). From early childhood to maturity, exercise intervention significantly reduced kisspeptin expression. From early childhood to maturity, high-fat diet intervention significantly reduced kisspeptin expression. Exercise intervention upregulated the decrease of kisspeptin expression caused by the high-fat diet, but the effect at maturity was not effective. As for GPR54, there was no significant change from early childhood to pre-puberty; then GPR54 expression increased significantly from pre-puberty to puberty, and from puberty to maturity the level remained relatively stable. Exercise intervention had no significant effect on the expression of GPR54 from early childhood to puberty, and then significantly downregulated the expression level of GPR54 in maturity. High-fat diet intervention upregulated the expression of GPR54 from early childhood to pre-puberty and significantly downregulated the expression level of GPR54 during maturity . Exercise intervention did not change the KISS-1 mRNA expression trend in rat testis, but decreased the expression level, especially in pre-puberty and puberty (p < 0.05). High-fat diet intervention significantly reduced the expression of KISS-1 mRNA in pre-puberty (p < 0.01), and then the expression of KISS-1 mRNA increased, but with no significant difference with the C group. Exercise intervention for rats fed with a high-fat diet changed the expression trend of KISS-1 mRNA. The expression trends of GPR54 mRNA and GPR54 protein were similar; both increased significantly from pre-puberty and puberty, and remained stable for the rest of the time (Table p < 0.01). Exercise intervention improved the expression of GPR54 mRNA caused by a high-fat diet, but there was no significant difference .Regression analysis of GSI and testosterone concentrations in testicular tissues of rats at different stages and with different intervention factors was conducted. Table p < 0.05), as Table Regression analysis of KISS-1/GPR54 system gene and protein expression levels in testicular tissues, and testicular tissue testosterone concentrations in different stages and with various intervention factors. A correlation existed only at a few special times between KISS-1/GPR54 expression in testis and testicular tissue testosterone concentration  PND 21st day ; (b) PND 35th day , (c) PND 43rd day , and (d) PND 56th day . The results showed that from early childhood to early adolescence, the gonads of male rats developed rapidly. The GSI and testicular tissue testosterone increased significantly; gonad development slowed down from pre-puberty and puberty, then accelerated again from puberty to maturity, and finally developed completely. With gonadal development, KISS-1 was expressed in spermatogenic cells and interstitial cells of rat testis, and the expression of KISS-1 in interstitial cells increased significantly. GPR54 first appeared in spermatogenic cells, then in testicular interstitial cells. The protein expression of KISS-1 and GPR54 in testicular tissues increased gradually. KISS-1 protein increased rapidly from puberty to maturity, and GPR54 protein increased rapidly from pre-puberty to puberty. The expression level of KISS-1 first increased and then stabilized to a peak at PND 35th day, while the expression level of GPR54 first increased and then decreased, and peaked at PND 43rd day.p < 0.05), mainly in interstitial cells [In this study, immunohistochemical results showed that the KISS-1/GPR54 system was expressed in spermatogenic cells and stromal cells in early childhood (21 D), but less in stromal cells, with low levels of protein and gene expression and low levels of testosterone. At this stage, the testicular supporting cells are still immature. At this time, the ability of phagocytosis is strong, which will inhibit the differentiation of spermatogonia. Also at this time, mesenchymal cells are at a low stage of development, and the number of mesenchymal cells in the field of vision is small. Anjum et al. found that kisspeptin was expressed in testes of newborn male Parkes mice, and the expression level decreased significantly from newborns (1 day old) to 4-weeks old  intervention on the expression of the KISS-1/GPR54 system in testis tissue of rats in the growth phase. Our study found that high-fat diet intervention resulted in a decrease in the testicular index from pre-puberty in rats; testicular tissue testosterone decreased significantly from prepuberty, and endocrine function was impaired; rat testicular tissue structure was loose, and the number of mature sperm decreased. The high-fat diet (45%) intervention had no significant effect on the localization of KISS-1/GPR54 in rat testis, but affected its expression in rat testis by downregulating the expression of KISS-1 protein.Related studies have shown that energy status and energy sources can affect pubertal initiation and gonadal development –21. Cui As mentioned above, energy status can affect puberty initiation , 23, 24.The energy state of the body affects the expression of KISS-1/GPR54. The negative energy balance pattern of exercise, cold exposure, and fasting is mainly concentrated in the hypothalamus for the expression of KISS-1/GPR54, and there are also inconsistent results. In energy-deficient rats and sheep, hypothalamic KISS-1 mRNA is decreased, LH levels decrease, and puberty stops, but puberty returns to normal after the injection of kisspeptin, LH and androgen levels also return to normal. Similarly for the hypothalamus, if the KISS-1 mRNA expression between AVPV and ARC is studied separately, different results will be found. Yamada et al. found that the level of KISS-1 in ARC was decreased by lactation stimulation in lactating rats . These sOur study found that exercise under standard diet did not alter the expression of KISS-1 in rat testis tissue but downregulated KISS-1 protein and gene expression levels in rat testis; exercise did not alter the expression trend of GPR54 gene and protein from early childhood to adolescence, but led to the decreased expression of GPR54 protein at maturity. Correlation analysis found that at the PND 35th day, the testicular tissue testosterone decline caused by exercise was significantly correlated with the KISS-1 protein expression level, indicating the involvement of KISS-1/GPR54 in prepubertal testis. When the system begins to participate in the secretion of sex hormones, the lack of energy due to exercise may affect the level of sex hormones mainly by affecting the expression level of KISS-1.On the other hand, this study found that exercise intervention can improve testicular development in rats with a high-fat diet, and can especially improve sex hormone disorders caused by high-fat diet. Under exercise intervention, the testicular tissue testosterone decline caused by a high-fat diet was improved. At the same time, the pre-puberty KISS-1/GPR54 system began to participate in the stage of sex hormone secretion, and exercise intervention upregulated the expression level of KISS-1/GPR54 at this stage. The specific mechanism leading to the downregulation of KISS-1 protein expression and gene expression after exercise intervention at maturity remains to be further studied.According to the results of the regression analysis and the changes in the expression of KISS-1/GPR54 in the testis, we found that the expression of kisspeptin in prepubertal testis increased in interstitial cells in addition to continued expression in spermatogonia, indicating that the KISS-1/GPR54 system in the testis continues to participate in testicular parenchymal development at this stage. It also may begin to participate in the secretion process of sex hormones.As mentioned above, kisspeptin may participate in puberty initiation in the ovary , 26, 27.Our study found that the high-fat diet caused both KISS-1 protein and gene expression levels in testis to be significantly lower than in the standard diet group, significantly downregulated the expression of GPR54 at maturity, and changed the KISS-1/GPR54 system expression trend. At the same time, correlation analysis showed that testosterone levels in rat testis were significantly correlated with GPR54 protein expression at 56D, indicating that the decrease of GPR54 protein expression induced by a high-fat diet may be the influencing factor of testosterone decrease in rat testis.In this study, we found that exercise intervention can improve testicular development under high-fat diet intervention by improving testicular tissue lipid droplet deposition and testicular tissue testosterone by means of morphology, testicular index, testicular tissue testosterone, and other indicators. The KISS-1/GPR54 system may be involved in improving androgen secretion, but the mechanism needs further study.The KISS-1/GPR54 system is expressed in rat testis during early childhood (21 D), prepuberty (35 D), puberty (43 D), and maturity (56 D). From early childhood to maturity, the expression of the KISS-1/GPR54 system increased and peaked at maturity (56 D).A high-fat diet can inhibit the testicular development of male rats in the growth phase, can downregulate the protein and gene expression levels of the KISS-1/GPR54 system in testis tissue, and can change the expression trend of the KISS-1/GPR54. Its role needs further study.2max moderate-intensity aerobic exercise will downregulate the expression level of testis tissue KISS-1/GPR54, change the inhibitory effect of a high-fat diet on testicular development in male rats, and upregulate KISS-1/GPR54 in the prepubertal stage. Whether KISS-1/GPR54 in testicular tissue participates in its regulation requires further study.60–70% VOAdditional file 1. Supplement 1 Body Weight of Growing Rats.Additional file 2. Supplement 2 Immunolocalization of KISS-1 in Rat Testis During Different Stages of Growing Rat (40X).


Drug-loaded, PEGylated, organic-modified silica (ORMOSIL) nanoparticles prepared by microemulsion condensation of vinyltriethoxysilane (VTES) were investigated as potential nanovectors for cancer therapy. To target cancer stem cells, anti-CD44v6 antibody and hyaluronic acid (HA) were conjugated to amine-functionalized PEGylated ORMOSIL nanoparticles through thiol-maleimide and amide coupling chemistries, respectively. Specific binding and uptake of conjugated nanoparticles were studied on cells overexpressing the CD44v6 receptor. Cytotoxicity was subsequently evaluated in the same cells after the uptake of the nanoparticles. Internalization of nanocarriers loaded with the anticancer drug 3N-cyclopropylmethyl-7-phenyl-pyrrolo- quinolinone (MG2477) into cells resulted in a substantial increase of the cytotoxicity with respect to the free formulation. Targeting with anti-CD44v6 antibodies or HA yielded nanoparticles with similar effectiveness, in their optimized formulation. The risk of developing a cancer has been constantly increasing during the last century. Today, about 16% of global deaths is due to cancer, making it the second leading cause of death .Therefore, the need for strategies aimed at increasing the accumulation of the drug at the target site increased the interest on nanoparticles ,4. TheseOn the other hand, active targeting consists in conjugating nanoparticles with ligands with high binding affinity for specific antigens overexpressed by cancer cells. In several studies this appears to be a more effective approach to ensure or enhance the selective accumulation of the nanosystems in the target tissue ,18,19,20An attractive target for cancer therapy is represented by cancer stem cells (CSCs). CSCs are tumor cells that have the principal properties of self-renewal, clonal tumor initiation capacity, and clonal long-term repopulation potential . AnotherAmong ligands overexpressed by CSCs, the CD44 receptor is a cell surface glycoprotein involved in several processes, including cell–cell interactions, cell adhesion, and migration ,27,28,29Moreover, CD44 is the major hyaluronan receptor ,37. HyalIn this work, we selected organic-modified silica (ORMOSIL) nanoparticles as vectors for CD44 targeting. Silica nanoparticles have been among the most studied nanomaterials for biomedical applications in the last decade ,47,48. TAmong anticancer agents, we selected a derivative of 7-phenylpyrrolo-quinolinone, which had previously demonstrated a significant cytotoxic activity in several cancer cell lines by acting as tubulin polymerization inhibitor . Moreove2000 layer, and conjugated with an anti-CD44v6 monoclonal antibody or with HA. Results demonstrated that these nanocarriers were selectively internalized by cells overexpressing the CD44v6 and significantly increase the cytotoxicity of the transported drug upon short incubation times.Herein, we report the design, synthesis and investigation of a CD44v6-targeting nanocarrier system based on multifunctional self-organized ORMOSIL nanoparticles. The nanoparticles were loaded with the MG2477 drug or with Rhodamine B silanized derivative, coated with a dense PEG® water purification system (Merck KGaA). Solvents were of analytical reagent grade, laboratory reagent grade or HPLC grade. Reactions were monitored by Thin Layer Chromatography (TLC) on 0.25 mm 60 F254 Merck silica gel plates (Merck KGaA) and, when necessary, stained with ninhydrin. ORMOSIL nanoparticles (NPs) and amine-functionalized ORMOSIL nanoparticles (NH2-NPs) were prepared according to previously reported procedures .,50.2-PEG2-NPs, PEG-Si and NH2-PEG-Si were added in a 10:1 ratio at the same total concentration of the Brij 35 surfactant (5 mM). Derivative NH2-PEG-Si ensures the presence in the coating of the primary amino groups needed for the conjugation with the targeting agents. The presence of the amino groups was confirmed with the fluorescamine test  was obtained by the thiol-maleimide chemistry, introducing maleimide moieties in the nanoparticles and thiol groups in the antibody  propionic acid N-hydroxysuccinimide ester (MBS). Purification was performed by ultrafiltration with membranes with a 10 kDa cut-off. The successful conversion of the amino groups of NH2-NPs in maleimide groups was verified with the fluorescamine test that confirmed the decrease of the amount of NH2 groups in the nanoparticles’ coating. The conjugation of the maleimide-NPs with the antibody was performed by incubating them in different ratios at RT for 48 h in PBS. Two different batches of conjugated NPs were prepared using 10-fold different amounts of antibody  and different ratios , zeta potential, fluorescence emission spectroscopy, thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) . DLS Ta and TEM 2-NPs have slightly negative Z-potential, suggesting that most of the surface is coated by PEG or vinyl groups, with only a few residual deprotonated silanol groups responsible for the small negative charge.The Z-potential of the NPs before conjugation  was −1 mV . Z-Poten2-NPs was calculated by fluorescence spectroscopy using a calibration curve built in a solvent mixture which could reproduce the position of the emission maximum, hence matching the internal polarity of the NPs. A concentration of 46.5 μM was obtained for a 4.30 mg/mL NH2-NPs suspension, corresponding to a 0.4% w/w loading.The concentration of the anti-cancer drug MG2477  loaded iConjugation with Ab-CD44v6 resulted only in a slight decrease of the NPs Z-potential, as expected on the basis of the typical isoelectric value of antibodies, which is close to neutrality. The average diameter measured with DLS remained almost unaffected, but the PDI values underwent a substantial increase, indicating the formation of a few aggregates in the sample. Indeed, after prolonged conservation of the samples at 4 °C the formation of sediments, which could be readily re-dispersed by sonication, was observed. TEM images confirmed, however, that the ORMOSIL cores remained unaffected and aggregation was not extensive.Protein conjugation could be demonstrated by SDS-PAGE analysis in reducing conditions, i.e., treatment with heat and β-mercaptoethanol. This caused the detachment of part of the antibody from the NPs, due to the breaking of the disulfide bridges, allowing the antibody to run along the polyacrylamide gel . In the 1x-Rho-NPs), was 0.03 mg/mL. This corresponded to a 3% w/w ratio. Approximate geometrical calculations indicated that about 15% of the particles surface was coated by the antibodies (~120 antibody molecules per NP). In the case of a 1 mg/mL suspension of MG2477-loaded NPs conjugated with the antibody at the highest amount (hereinafter referred to as Ab-CD44v610x-MG2477-NPs), a 0.162 mg/mL concentration of antibody was obtained. This corresponded to a 16% w/w ratio (~640 antibody molecules per NP) and an almost total surface coverage (86%).Titration of conjugated antibody was performed as described in −1), the NH bending (1600–1400 cm−1), CC and CO stretching (1200–1000 cm−1), and SiO stretching (1000–800 cm−1). After conjugation with HA fragments, the spectrum was very close to that of the unconjugated particles but for a few signals in the 1800–1200 cm−1 regions, where C=O stretching signals from carboxyl (1743 cm−1) and amide residues  were detected. Noticeably, the presence of the amide signals clearly confirmed the effective conjugation of the biopolymer to the NPs  were used for the in vitro experiments when not otherwise stated. Binding of NPs to cells was analyzed by incubating increasing concentrations of NPs, loaded with Rho, and conjugated with Ab-CD44v6 (Ab-CD44v6-Rho-NPs), with HEK-293A cells overexpressing CD44v6 (HEK-293A-CD44v6), at RT for 1 h, to allow in such a short incubation time only the binding of NPs to cells without internalization. After washing out unbounded NPs multiple times and fixing cells, Rho signal of bonded NPs was analyzed at a fluorescence microscope A. FluoreSelective internalization and intracellular distribution of NPs in HEK-293A-CD44v6 cells was analyzed by incubating the cells with different concentrations of Ab-CD44v6-Rho-NPs, Alb-Rho-NPs and unconjugated NPs (Rho-NPs), at 37 °C for 4 h, to allow entry of NPs in cells. Cells were then washed multiple times in order to remove any unbound NP and Rho signal was analyzed by fluorescence and confocal microscopy. The Rho signal was distributed in both nuclei and cytoplasm and was dose-dependent B. On theAdditional control experiments, performed using both CT-26 cells (expressing the CD44v6) and bovine cells 5050 (not expressing the CD44v6), showed that Ab-CD44v6-Rho-NPs can be efficiently internalized only in cells expressing CD44v6 . Similar results were obtained by using NPs conjugated with HA 22.5 kDa (HA-Rho-NPs). While unconjugated Rho-NPs did not show any internalization into cells, a strong Rho fluorescence signals was observed in samples treated with conjugated NPs, both by fluorescence  and Z stMG2477 is a potent growth inhibitor of several cancer cell lines in vitro. It acts by inhibiting tubulin polymerization and causes cells to arrest in metaphase ,42. CellTo investigate the effectiveness of targeted drug delivery with the NPs prepared, conjugated and unconjugated MG2477-loaded NPs, as well as the free drug, were incubated with HeLa-CD44v6 cells at the concentration of 0.1 µM (unless otherwise stated) for 4 h at 37 °C, then the cells were washed and allowed to grow for further 72 h in a renewed medium before MTT assay was applied. When Ab-CD44v6-MG2477-NPs were tested at either 0.01 or 0.1 µM , a cell viability reduction in the range of 20–40% was observed C. 10x-MG2477-NPs, antibody amount 10×) produced a lower cytotoxic effect compared to those conjugated with a lower amount . In the Ab-CD44v610x-MG2477-NPs, the nanoparticle surface is almost completely covered by antibody molecules (see earlier) and such a crowding could reduce the accessibility to the binding sites of CD44 [1x-MG2477-NPs was significantly greater compared to unconjugated MG2477-NPs , confirming the receptor-mediated internalization of the NPs into target cells. No significant differences were observed by increasing the drug concentrations from 0.01 to 0.1 µM  was observed for HA1x-MG2447-NPs and HA10x-MG2447-NPs, respectively, compared to unconjugated MG2477-NPs, confirming the specific HA-mediated internalization of NPs in target cells. Hence, a significant difference was observed between NPs conjugated with the different amounts of HA. On the opposite to what obtained with the antibody-conjugated NPs, a higher amount of conjugated HA (HA 10×) induced a higher cytotoxicity (p < 0.01) compared to the NPs conjugated with a lower amount. Being a linear polymer, HA likely is less sensitive to steric effects than Ab. In addition, no dependence from the molecular weight of the HA was observed, suggesting that the interaction with CD44v6 occurs with a small fragment of the biopolymer. Cell viability was also evaluated for the NPs (loaded with MG2447 drug at 0.1 µM) conjugated with HA with two different molecular weights (11.5 and 22.5 kDa) and in two different amounts (1× and 10×) and compared to unconjugated NPs D. A signp < 0.01) reduction of cytotoxicity was observed in cells treated with an excess of HA , subsequently washed, and then incubated with HA-MG2447-NPs for 4 h. At 72 h post-treatment, cells were analyzed by the MTT assay. A significant  at which the free drug was almost ineffective. In other words, the use of these targeted nanovectors resulted in the enhancement of the activity of the drug. The reason for such an effect probably lays in the change of the drug availability. In the free from, MG2477 is poorly soluble in water and, for this reason, accumulates slowly in cells. This, in part, explains the need for long incubation times to observe relevant viability reduction. Targeted NPs provide a much more efficient uptake pathway. They encapsulate the drug, preventing its aggregation and precipitation, and rapidly deliver it in the cell by receptor mediated uptake. In agreement with this hypothesis, we observed that unconjugated NPs reduced the cytotoxicity of the drug, likely by preventing its internalization in the cells. The results obtained provide also relevant comparative information about the effectiveness of different targeting strategies. Indeed, similar cytotoxicity was obtained with the Ab-CD44v6 and HA-conjugated NPs, in their optimized formulation. From this perspective, the use of the polysaccharide appears more interesting. First, the conjugation protocol is simpler; there is no need to pre-functionalize both the NPs and the targeting agent. Second, the targeting agent itself is less expensive and easier to produce in large amounts. Third, greater NPs and, consequently, drug concentrations are obtained when NPs are targeted with HA with respect to the antibody.Effectiveness of HA as a targeting agent appeared to be independent from its molecular weight, with the exception of small oligomers which proved to be ineffective (data not shown). Active targeting of the CD44v6 receptor was confirmed by the observation that a high concentration of free HA reduced the cytotoxicity. On the other hand, cytotoxicity was proportional to the amount of targeting agent conjugated to the NPs and high concentrations of the free targeting agent did not suppress the biological activity of the NPs. This suggests a relatively low affinity of HA for the CD44 receptors, which could have been enhanced by the NPs multivalency, or the need for the receptor clustering to promote the internalization, which could have been favored by the presence of a high number of targeting molecules on the NPs surface. In the case of the antibody-conjugated NPs, however, an inverse trend was observed, with NPs conjugated with a smaller amount of targeting agent being more effective than the ones conjugated with a larger amount of antibody. This observation is quite relevant as it confirms earlier observations that small amounts of antibodies are sufficient to obtain a good targeting and delivery efficiency ,35. When


Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a “cold-chain” is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315–375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability. Newcastle disease virus (NDV) is a highly contagious and important cause of loss in the poultry industry . It is aMost NDV strains are thermolabile and lose infectivity upon exposure to 50–55°C for 30 min. However, heat-resistant strains (such as V4) can maintain their hemagglutinin (HA) activity and infectious ability at 56°C for at least 30 min . PreviouFor the molecular characterization of thermostable NDV, the genome of several thermostable strains have been sequenced. By analyzing the sequences of different thermostable strains, In this study, we generated chimeric NDVs by exchanging viral F and/or HN genes between the heat-resistant strain HR09 (genotype VIII) and thermolabile strain La Sota. The differences in thermostability among these chimeric viruses were evaluated to identify thermostable determinants of NDV. Our results showed that the HN gene is a crucial determinant for NDV thermostability. Furthermore, we further performed a molecular dynamic simulation to analyze the stability of the HN protein structure at different temperatures and compared the amino acid sequences of thermostable and thermolabile viruses. We then investigated the influence of amino acid substitutions at positions 315, 369, and 329 on viral thermostability. The results showed that the mutations of S315P and I369V in the HN protein significantly enhanced the viral thermostability.The NDV heat-resistant strain HR09 was isolated from chickens and stored in our laboratory . The NDVThe full-length infectious cDNA clones (ICs) of the NDV heat-resistant strain HR09 and thermolabile strain La Sota, named cHR and cLa, respectively, were generated in plasmid TVT-7R (0.0)  from sixThree chimeric ICs, cHR-La-F, cHR-La-HN, and cHR-La-F/HN, were constructed by replacing the open reading frames (ORFs) of the F and/or HN genes in the cHR strain with the counterpart fragments from the La Sota strain . BrieflyTo analyze the amino acid sequence differences in the HN proteins of the thermostable NDV strains and La Sota strain, the HN gene sequences of currently available thermostable NDV strains were retrieved from GenBank. The amino acid sequences were aligned by Clustal W, and the results were analyzed by BioEdit software.1. The HN protein structure was visualized and analyzed with VMD software .To further investigate the possible mechanism of improved viral thermostability, the 3D structure of the HN protein was modeled by SWISS-MODEL software online+ and Cl– were added to neutralize the system. The HN protein was solvated in a cubic box consisting of the TIP3P water model, and the distance of protein atoms from the wall was more than 10 Å. The whole system was minimized using the descent method (10000 steps). A time step of 50 ps was used to balance the conformation before performing the MD simulation. By calculating the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values for backbone atoms at temperatures of 310 K (approximately 37°C) and 330 K (approximately 57°C), respectively, thermally sensitive regions of the HN protein were identified.The crystal structures of the HN protein of the La Sota strain were obtained from the Protein Data Bank (PDB ID code: 3t1e.1.A) and analyzed by molecular dynamics (MD) simulation, and potentially flexible regions in the HN protein were identified. The MD simulations were carried out by NAMD software  with a CTo investigate whether the three amino acid substitutions at the thermally sensitive regions of the HN protein affect viral thermostability, the full-length ICs of thermolabile cHR-La-HN were used as the backbone, and chimeric ICs containing substitution of single, double or triple amino acids at positions 315, 369, and 329 were constructed. Briefly, the mutations of S315P, I369V, and V329A generated on plasmids pLa-HN using PCR-based site-directed mutagenesis with three pairs of primers . FinallyRescuing of chimeric and mutant viruses was performed by cotransfecting the full-length ICs with three supporting plasmids  into BSR T7/5 cell lines using Lipofectamine 3000 , as previously described . Briefly50) assay (6 CEFs per well were infected at a multiplicity of infection (MOI) of 0.01 for 2 h, then washed three times with phosphate-buffer saline (PBS), and overlaid with medium containing 2% FBS and 2 μg/mL TPCK-treated trypsin . The infected cell supernatants were harvested at the indicated time points, and the virus was titrated using the 50% tissue culture infective dose (TCID50) assay according to the method of The pathogenicity of the rescued NDVs in 10-day-old SPF chicken embryos was determined by conducting the mean death time (MDT) assay . The tit0) assay . The gro50 assay and the HA test, respectively. HA assay was performed by micro-hemagglutination procedure using microtiter method. Briefly, the viruses were two-fold serially diluted with PBS, and then equal volumes of fresh 1% (V/V) chicken red blood cells were added and incubated at 37°C for 30 min. The HA titers of the viruses were determined as the highest dilution giving 100% agglutination. The decreased infectivity and HA titers of these viruses exhibited a logarithmic trend as the heat-treatment time increased. The thermostable and thermolabile viruses were examined at different time points by the heat-treatment assay.One milliliter of allantoic fluids infected with NDV were submerged into a water bath at 56°C, and transferred to an ice-cold water bath at the indicated time points to stop the heat treatment . The infThe NA activity of viruses was determined by a fluorescence-based assay using a Neuraminidase Assay Kit  according to the manufacturer’s instructions. Briefly, 70 μL of detection buffer and 10 μL of allantoic fluid infected with NDVs were added to 96-well polystyrene plates, followed by the addition of 10 μL Milli-Q water. The solution was then mixed gently for 2 min. Then, 10 μL of NA fluorescent substrates was added to the solution and mixed well. After incubation at 37°C for 30 min, the fluorescence with an excitation wavelength of 322 nm and an emission wavelength of 450 nm was measured by a fluorescence spectrophotometer . NA activity is shown as the fluorescence intensity of the samples minus the background values of non-infected allantoic fluid.5 TCID50/mL of NDVs were heat-treated at 56°C for 10 min, and then infected with Vero cells for 2 h at 37°C. Then the infected cells supernatant was discarded and kept in the DMEM (Gibco) supplemented with 2% FBS. At 36 h post infection, cell monolayers were washed with PBS for three times and fixed with pre-cooled methanol for 15 min. The cell monolayers were stained with Giemsa solution for syncytia observation under an inverted microscope. Syncytia are cells with three or more nuclei. Fusion activity was calculated from the fusion index, which involved calculating the ratio of the total number of nuclei to the number of cells in which these nuclei were observed. The value obtained from fusion index for the NDVs was expressed as the percentages of the value for the HR09 virus, which was considered to be 100%.The fusion activities of the NDVs were examined as previously described . Brieflyt-test. A value of p < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism Software Version 5.0 . Measured values are expressed as the mean with standard deviation (SD). Statistically significant differences between different groups were assessed using a The role of the F and HN genes in the thermostability of NDV was investigated. Six ICs of chimeric NDVs were constructed; the ORFs of F and/or HN were exchanged between the ICs of the heat-resistant strain HR09 and the thermolabile strain La Sota . The chi50/0.1 mL values of chimeric viruses except cLa-HR-F were almost more than 8.0 log10. The growth kinetics of parental and chimeric NDVs were evaluated in CEFs (p > 0.05).The pathogenicity of chimeric viruses was investigated. The chimeric NDVs were performed by MDT assay in 10-day-old SPF chicken embryos . Of the  in CEFs . The par50 assay , but significant higher than those of HR09 (p < 0.05). In addition, there were no significant differences in virus titers between the mutant viruses and chimeric cHR-La-HN; and the chicken embryos infectivity titers were ≥8.3 log10 EID50/0.1 mL, and the HA titer was 10.0 log2, respectively , with a titer loss of 4.0 log10. Among the three mutant viruses, the titer loss of cHR-La-HN-A was higher than that of cHR-La-HN-B and cHR-La-HN-C within 20 min (p < 0.05). Additionally, there was no statistically significant difference in the titer loss between cHR-La-HN-B and cHR-La-HN-C at the same heat treatment time.To investigate the difference in thermostability between chimeric virus and mutant viruses, heat resistance was evaluated by performing an EID at 56°C . Within 2 in 5 min, whereas the HA titer of three mutant viruses decreased approximately by 3.0 log2 at the same time. In addition, the HA titer loss of cHR-La-HN-A was much greater than that of cHR-La-HN-B and cHR-La-HN-C at 15 min (p < 0.05). There was no statistically significant difference in the HA titer loss between cHR-La-HN-B and cHR-La-HN-C at the same heat treatment time (p < 0.01). Moreover, the NA activity of cHR-La-HN-B was similar to that of cHR-La-HN-C, and the activities of both cHR-La-HN-B and cHR-La-HN-C were much higher than that of cHR-La-HN-A . These results suggested that when the HN gene of thermostable HR09 was replaced by thermolabile La Sota, the fusion activity of chimeric virus have been decreased rapidly after heat treatment at 56°C. The mutant viruses possessed higher fusion activity than the chimeric virus under the same heat treatment condition.In order to explore the effects of the mutant viruses and chimeric virus on their fusogenic activity after heat treatment at 56°C, the syncytium formation was observed and fusion index assay was performed in Vero cells . The HR0To analyze the location of the amino acids at positions 315, 329, and 369, the structure of the HN protein of the La Sota strain was analyzed, and the locations of Ser315, Val329, and Ile369 are indicated in p < 0.05). Although, it is well-known that the HN gene is responsible for the replication and pathogenicity of NDV, the role of the HN gene in viral thermostability has not been well understood. Sequence alignment of the HN protein from different thermostable strains showed that the HN proteins of genotype I strains V4 and I-2 have 616 and 578 amino acids, respectively, whereas the HN protein of the HR09 strain has 572 amino acids, which is less than that of AF2240-I (583 amino acids)  . WhetherMD simulation has widely been used to explore the thermal-stable mechanism of industry enzymes . By calcThe sialic acid binding sites of the HN protein play a key role in the attachment of viruses to cells and are usually conserved across isolates irrespective of their thermostability phenotypes . ResidueIn summary, our study reveals that the HN protein is the major determinant of NDV thermostable. Furthermore, the mutations of S315P and I369V in the HN protein could contribute to enhancing viral thermostability and HA and NA activity. These findings have important implications for understanding the thermostable mechanism of NDV and play a positive role in thermostable NDV vaccine development.https://www.ncbi.nlm.nih.gov/genbank/, JX524203.1; https://www.ncbi.nlm.nih.gov/genbank/, AY935499.2; https://www.ncbi.nlm.nih.gov/genbank/, JX443519; https://www.ncbi.nlm.nih.gov/genbank/, MF285077; https://www.ncbi.nlm.nih.gov/genbank/, JX012096.2; https://www.ncbi.nlm.nih.gov/genbank/, KC844235.1.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: YC, XZ, and YW carried out the study design. BR, CM, and PD performed the experiment and data analysis. BR wrote the manuscript. MG, CZ, and YW contributed to the revision of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer’s disease (AD) is a destructive and burdensome neurodegenerative disease, one of the most common characteristics of which are neurofibrillary tangles (NFTs) that are composed of abnormal tau protein. Animal studies have suggested that dl-3-n-butylphthalide (dl-NBP) alleviates cognitive impairment in mouse models of APP/PS1 and SAMP8. However, the underlying mechanisms related to this remain unclear. In this study, we examined the effects of dl-NBP on learning and memory in P301S transgenic mice, which carry the human tau gene with the P301S mutation. We found that dl-NBP supplementation effectively improved behavioral deficits and rescued synaptic loss in P301S tau transgenic mice, compared with vehicle-treated P301S mice. Furthermore, we also found that it markedly inhibited the hyperphosphorylated tau at the Ser262 site and decreased the activity of MARK4, which was associated with tau at the Ser262 site. Finally, dl-NBP treatment exerted anti-inflammatory effects and reduced inflammatory responses in P301S mice. In conclusion, our results provide evidence that dl-NBP has a promising potential for the therapy of tauopathies, including AD. Alzheimer’s disease (AD), the foremost cause of dementia in the elderly, is a complex multi-factorial neurodegenerative disease. It is characterized by two classic neuropathological hallmarks: senile plaques consisting of a progressive accumulation of amyloid-β peptide (Aβ) and neurofibrillary tangles (NFTs). NFTs are formed by the aggregation of paired helical filaments (PHFs) composed of truncated and hyperphosphorylated microtubule-associated protein tau . The latDl-3-n-butylphthalide (dl-NBP) was first isolated from the seeds of Chinese celery. It is a synthetic chiral compound that contains the L- and D-isomers of butylphthalide. In 2002, this compound was approved for use with stroke patients by the State Food and Drug Administration of China. Some evidence has revealed that the administration of dl-NBP ameliorates oxidative stress and pro-mitochondrial function, decreases neuronal apoptosis, and inhibits the inflammatory responses in experimental ischemic models . FurtherHerein, we studied the role of dl-NBP in an AD transgenic mouse model for AD-related tauopathy, which expresses the human tau gene with the P301S mutation. We found that the administration of dl-NBP significantly decreased the levels of hyperphosphorylated tau at Ser262 in P301S mice, compared with vehicle-controlled mice. Furthermore, treatment with dl-NBP significantly improved synaptic integrity, reduced neuroinflammation, and ultimately ameliorated cognitive impairment in P301S mice, compared with vehicle-treated P301S mice.∗P301S)/Nju] that express human mutant microtubule-associated protein tau . Dl-NBP was used for oral management at 30 mg/kg body weight, as in previous studies . Eleven For spatial learning and memory measurement, we performed the Morris water maze (MWM) test . For theWe performed an open-field test (OFT) in a square box (45 × 45 cm). The environment was kept quiet. Before the test began, each mouse was allowed to adapt to the open-field arena for 30 min. Then, the mice were placed in the center of the box. A computer-assisted infrared tracking system recorded the mouse for 5 min. The times spent in the center and peripheral zones were recorded. In addition, the total distance traveled was used to measure locomotor activity. To avoid the influence of odor in the wooden box, the box was wiped with a solution of 70% ethanol.Mice were placed in a square box  that included a metal floor and a sound system. First, a test mouse was placed in the box for 3 min to adapt to the environment. Then, during the training session, the mouse was given an auditory cue of 70 dB for 30 s, followed by a mild foot shock (0.5 mA for 2 s). The operation was repeated twice with 2 min intervals. For the short-term memory test, the mice were placed in the chamber again without stimulus for 5 min after 4 h of the training session, and the “freezing” time was recorded for a memory assessment. For the 24 h long-term memory test, the process was repeated. A camera and a circuit board were connected to a personal computer, and software  controlled the circuit and recorded data.2PO4, 26 mM NaHCO3, 2 mM MgSO4, 2.5 mM CaCl2, and 10 mM glucose . Coronal brain slices (300 μm thick) were cut at 4°C in the aCSF using a Leica VT1000S vibratome  and saturated with carbogen (95% O2 and 5% CO2). After slicing, sections were immediately transferred to an incubation chamber filled with oxygenated aCSF and were equilibrated at room temperature for 30 min before recording. Slices were placed in a chamber and recorded with an 8 × 8 microelectrode array at the bottom  and kept submerged in aCSF. Signals were detected using a MED64 System . We recorded the field excitatory postsynaptic potentials (fEPSPs) in the CA1 region by stimulating CA3 neurons. We used the current that corresponded to a maximum amplitude of 40% of the induced fEPSPs as the appropriate electrical stimulation. The baseline was recorded for 30 min, followed by high-frequency stimulation . Then, long-term potentiation (LTP) data continued to be recorded for 1 h. The magnitudes of LTP were quantified as the mean percentage of baseline fEPSP initial slope.Mice were decapitated after being deeply anesthetized. The brains of the mice were removed and put into oxygenated ice-cold artificial cerebrospinal fluid (aCSF) that was composed of 124 mM NaCl, 2 mM KCl, 1.25 mM NaHAfter the behavioral test, half of the mice were randomly selected from the three groups and anesthetized. The cortexes and hippocampus of these mice were removed and stored at −80°C for biochemical studies. Protein samples were extracted, and their concentrations were measured. The protein samples were separated on a 10 or 12% SDS-PAGE gel. Then, they were transferred onto polyvinylidene fluoride (PVDF) membranes or nitrocellulose (NC) membranes, followed by blocking in 5% non-fat milk for 1.5 h at room temperature. The membranes were then incubated with primary antibody  at 4°C oAfter the behavioral test, half of the mice were randomly selected from the three groups and anesthetized. Then, they were perfused with sodium chloride and paraformaldehyde. After, their brains were removed, immersed in paraformaldehyde, and embedded in paraffin. Brain slices were cut coronally at a thickness of 5 μm. The slides were dewaxed, and antigen was repaired (high temperature and high pressure conditions were used to repair the tissue section) and incubated with the corresponding primary antibody  overnighBrain slices were deparaffinized in xylene and rehydrated in ethanol. These were successively passed in different concentrations of ethanol of 100, 95, 90, and 70%, each for 5 min. The brain slices were then stained with Nissl solution for 3 min and cleaned with distilled water for 10 s. This was followed by passing in dimethylbenzene for 5 min. The brain slices were then sealed with neutral balsam. Finally, images were collected using an Olympus photomicroscope .P < 0.05. Meanwhile, comparing P301S with dl-NBP-treated P301S mice, results were considered to have significant differences at P < 0.05.All statistical results are expressed as means ± SEMs. We employed one-way and two-way ANOVAs for comparisons between the three groups. Data were analyzed using GraphPad Prism 7.0 software. Comparing P301S with WT mice, results were considered to have significant differences at Mice were treated with either dl-NBP or vegetable oil at 4 months old for 4 months. To evaluate whether dl-NBP treatment could ameliorate behavioral deficits in P301S mice, we conducted a MWM test, OFT, and fear conditioning test. The experimental methods involved in our study are described above. The MWM test is one of the most widely recognized behavioral tests used to assess hippocampal-dependent spatial learning and memory. Spatial learning was assessed using escape latency (time spent to find the hidden platform). As shown in In summary, the above results provide evidence that treatment with dl-NBP can effectively improve learning and spatial memory in P301S mice.Elevated levels of hyperphosphorylated tau are a major pathologic feature in AD. Thus, we conducted western blotting and immunohistochemistry to investigate whether dl-NBP treatment could decrease hyperphosphorylated tau in P301S mice. As shown in Taken together, these results showed that dl-NBP treatment evidently reduced tau hyperphosphorylation at Ser262 in the hippocampi of P301S mice.D-aspartate receptors (NMDARs) play an important role in shaping synaptic responses acutely and in the induction of LTP. Hence, we examined the protein levels of the NMDAR obligatory subunit NR2B and the AMPAR subunit GluR1. Post-synaptic density protein 95 (PSD95) is also a critical synaptic protein. It is important for synaptic plasticity. Its level is diminished in AD. The data showed that protein levels of the synaptic proteins  in the hippocampus were lower in P301S mice than in WT mice. Interestingly, the protein levels were elevated in P301S mice treated with dl-NBP, compared with vehicle-treated P301S mice. Nissl staining is a method of dyeing nerve tissue with basic dyes. As shown in To examine whether dl-NBP helped improve the synaptic function of P301S mice, we assessed the levels of several synapse-related proteins . α-AminoSynaptic plasticity is the premise of learning and memory. One of its primary manifestations is LTP. As we wondered whether dl-NBP improved synaptic plasticity in P301S mice, we utilized acute brain slices from three groups of mice and electrorecorded hippocampal slices to examine the LTP of the CA3-to-CA1 region . FEPSPs Hence, the results suggested that dl-NBP has the potential to rescue the impairment in synaptic function in P301S mice.As GSK3β, CDK5, PP2A, and MARK4 have been linked to tau hyperphosphorylation, we next explored the expression of these kinases and phosphatases after treatment with dl-NBP. GSK-3β increases p-tau levels. Phosphorylation of GSK-3β at Ser9 decreased GSK-3β activity. PP2A helped to dephosphorylate tau. The phosphorylation of PP2A at Tyr307 was found to inactivate PP2A and increase hyperphosphorylated tau, finally causing NFTs. As shown in To assess whether dl-NBP helped reduce neuroinflammation in P301S mice, we next measured the levels of inflammatory markers. As shown in Taken together, these results demonstrate that the administration of dl-NBP reduces neuroinflammation in P301S mice.AD is the leading cause of dementia among the elderly. As clinical trials that target anti-Aβ therapies have failed to inhibit the progression of AD patients’ cognitive decline, tau has been considered to have a good prospect as a therapeutic target. As a drug for stroke treatment in China, dl-NBP has been reported to ameliorate cognitive impairment in vascular dementia. In addition, it has been shown to significantly improve cognitive functioning in AD models that target Aβ. The P301S mouse, which carries the human tau gene with the P301S mutation, is a well-characterized mouse model that is used to investigate effective treatment strategies for tauopathies. This study aimed to investigate the potential protective role of dl-NBP against cognitive decline in P301S transgenic mice.We demonstrated that chronic gastric perfusion for 16 weeks of dl-NBP reduced cognitive deficits in P301S mice. In the MWM behavioral test, we observed that treatment with dl-NBP remarkably ameliorated the spatial learning and memory impairments in P301S mice, as deduced by the shorter escape latencies observed in the training phase and the number of entries into the platform zone during the probe trial. Moreover, as a second measure to assess memory, the contextual fear conditioning test revealed that dl-NBP treatment improved long-term memory, as shown by an obviously increased freezing time during the memory test at 24 h. As learning and memory are regulated by synaptic function and integrity, our behavioral test results suggest that treatment with dl-NBP could play a protective role for synaptic deficiency in P301S mice.Synaptic plasticity is a functional term that describes alterations in synaptic efficiency. It is thought to be the biological foundation of learning and memory. Cognitive decline among the aged is considered to be related to a decrease in structural and functional plasticity in the hippocampi. LTP is an important form of synaptic plasticity . We examThe accumulation of hyperphosphorylated tau is evident in P301S mice. We explored whether dl-NBP affects the regulation of hyperphosphorylated tau. In previous studies, hyperphosphorylated tau had been found in more than 40 serine/threonine residues from various neurodegeneration patients . We seleConsidering the crucial impact of tau kinases and tau phosphatases on the abnormal hyperphosphorylation of tau in the brains of patients with AD, we further examined the protein levels of tau kinases and tau phosphatases. Microtubule affinity-regulating kinase (MARK) , glycogeNeuroinflammation has also been reported to play a significant role in AD . In addiOur study provides experimental support for the protective effect of dl-NBP in P301S mice. The administration of dl-NBP markedly reduced tau phosphorylation levels at Ser262, improved memory and learning, and ameliorated synaptic deficits in AD mice. In addition, treatment with dl-NBP also decreased the inflammatory response in AD mice. Taken together, these new findings suggest the potential of dl-NBP as a treatment option for AD and other tauopathies.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology.GL designed the study. YC, YY, and JH carried out the experiments and performed the measurement. YW, XJ, and LL analyzed the data. YC drafted the manuscript. GL, RM, and ZW modified the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Temperature-responsive photonic coatings are appealing for a variety of applications, including smart windows. However, the fabrication of such reflective polymer coatings remains a challenge. In this work, we report the development of a temperature-responsive, infrared-reflective coating consisting of a polymer-stabilized cholesteric liquid crystal siloxane, applied by a simple bar coating method. First, a side-chain liquid crystal oligosiloxane containing acrylate, chiral and mesogenic moieties was successfully synthesized via multiple steps, including preparing precursors, hydrosilylation, deprotection, and esterification reactions. Products of all the steps were fully characterized revealing a chain extension during the deprotection step. Subsequently, the photonic coating was fabricated by bar-coating the cholesteric liquid crystal oligomer on glass, using a mediator liquid crystalline molecule. After the UV-curing and removal of the mediator, a transparent IR reflective polymer-stabilized cholesteric liquid crystal coating was obtained. Notably, this fully cured, partially crosslinked transparent polymer coating retained temperature responsiveness due to the presence of non-reactive liquid-crystal oligosiloxanes. Upon increasing the temperature from room temperature, the polymer-stabilized cholesteric liquid crystal coating showed a continuous blue-shift of the reflection band from 1400 nm to 800 nm, and the shift was fully reversible. Stimuli-responsive photonic polymer coatings are able to change color in response to an external stimulus . These “Polymeric materials are required for stable-performance photonic coatings. Photonic coatings of temperature-responsive non-polymerized Ch-LC mixtures encapsulated in polymer protective layers were recently reported, showing a good stability and bandshift range by the Sm-Ch pre-transitional effect, but a subtle stratification process is required for their fabrication ,18,19. AIn our previous work, we prepared a temperature-responsive coating by blade-coating a mixture of oligomeric polysiloxane and a chiral dopant . This no1H NMR and 13C NMR (−1 in E7 (by wt%). M3 contains a protecting group which was used to prepare the acrylate-containing mesogenic group after the hydrosilylation reaction. It was used instead of the final mesogenic group to avoid participation of the acrylate group during the hydrosilylation.We designed a side-chain polysiloxane LC terpolymer with both cholesteric and smectic LC phases and that is crosslinkable TP, . This po 13C NMR . Althougavg) of 3.9. The polymer backbone was determined to be short in order to obtain only a small mol% of crosslink-able oligomers, which also maintained a low crosslinked ratio in the coatings. In the first synthesis step, TP-s1 was made through a hydrosilylation reaction of M1, M2, and M3 with PMHS, in a yield of 77.6%. The product TP-s1 was found to have a navg of approximately 6, calculated based on the 1H NMR spectrum -protecting group was removed in the presence of pyridinium p-toluenesulphonate and ethanol to form TP-s2. This deprotection reaction that formed the free phenolic group was completed without affecting the other mesogen side groups (see in 1H NMR in W) of TP-s2 had increased by 42% compared to TP-s1. Furthermore, a wider molecular weight distribution (polydispersity increased from 1.33 to 1.40) was observed. This molecular weight increase was disproportional to the weight loss of this step (yield = 90%). Moreover, a considerable number of cyclic oligomers was found in the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) spectrum, which was absent in TP-s1  (OMHS) in three steps of synthesis . M1 reprin TP-s1 . Among tof TP-s2 . Based o1H NMR spectrum of TP . Therefore, a rather high network fraction after crosslinking is expected.The third reaction step, the esterification of the phenol groups in TP-s2 with carboxylic acid building block 5 using N,N′-dicyclohexylcarbodiimide (DCC) and N,N-4-dimethylaminopyridine , yielded the desired terpolymer (TP). The um of TP  confirmeum of TP . MALDI-Tum of TP  confirmeThe liquid crystalline properties of the TP were subsequently examined using differential scanning calorimetry (DSC). Despite being a complex mixture of various species, TP shows clear peaks of phase transitions on DSC curves a. Isotro1H NMR spectrum . Other materials were lab-synthesized. For details of the syntheses, see the The TP and CM were weighed in brown glass vials, in a weight ratio of 3:1. Subsequently, initiator 184 was added via solution in a volumetric flask (2 mg/mL in unstabilized THF), precisely in 0.25 wt% or 0.5 wt% to the weight of TP. More unstabilized THF was then added to dissolve all components, leading to a solution of ~45 wt% in the end.The same procedure was followed as in .2 for 10 min. After the exposure, the coatings were kept in nitrogen atmosphere for 1h, and were cooled down to room temperature. CM component was removed from the coatings completely, by dipping the coatings in heptane for at least 16 h, with an exchange of solvent three times. The coatings were then collected and dried in air.An RK PrintCoat Instruments K control coater was used to prepare the coatings. The mixtures were applied on polyimide-rubbed glass substrates and placed at 60 °C for 1 h to evaporate the solvent. Then, the coatings were applied using a 30 µm gap , which was pushed forward over the mixture automatically by the coater. The temperature of the substrate surface during the application was approximately 51 °C. The speed of the applicator movement was 0.5 cm/s. The coatings were then cooled down to 43–47 °C at the surface, flushed with nitrogen constantly for 1.5 h, and illuminated with UVA using an EXFO Omnicure S2000 mercury lamp, at an intensity of 20 mW/cmThe thickness of the obtained coatings was measured using a Fogale Zoomsurf 3D interferometer. The coatings fabricated for optical measurements were ~11–13 μm in thickness.For the control experiment of the mixture without using initiator 184, the coating was cooled to room temperature right after the coating application without UVA curing.For the samples used in the crosslink fraction tests, the mixtures were coated on 9 × 12 cm clean glass substrates without polyimide layers. A larger portion of materials was used to create larger coating samples.29Si measurements, Chromium(III) acetylacetonate and tetramethylsilane were added to the sample, and the measurements were performed under the inverse-grated mode. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Bruker Autoflex Speed MALDI-MS instrument, with a DCTB as matrix agent. Differential scanning calorimetry (DSC) curves were measured with a DSC Q2000, TA Instruments. Gel penetration chromatography (GPC) was performed on a Shimadzu LC-2030C.3D instrument equipped with a PDA-254 nm detector, using chloroform as the eluent and monodisperse polystyrene calibration standards. Polarizer microscopy (POM) images were taken on a Leica microscope DM2700 in transmission mode, equipped with polarizers.NMR spectra were recorded on a 400 MHz Bruker Avance III HD spectrometer. For The coatings were collected in 0.2 μm PTFE filters and weighed to find approximately 20 mg of materials on average. The filters containing these materials were flushed with a continuous flow of dichloromethane by syringes at room temperature, and then dried in a 40 °C oven. The flush-dry cycle was run until the weight of the dried filters did not reduce anymore. The crosslink fractions were calculated from the weight of the material left in the filter divided by the initial weight of material in the filter. The eluents were collected and analyzed with proton NMR and GPC.Transmission spectra of the coatings were measured on a Shimadzu UV-3102 PC UV/Vis/NIR spectrophotometer equipped with a MPC-3100 sample compartment and a Linkam TMS93 temperature controller. A polyimide coated glass substrate was used as baseline. The actual temperature of the coating was calibrated using a thermometer probe (Comark KM340). A scan of spectrum started exactly every 5 min when the coating was kept at one temperature, until the new spectrum was identical to the previous one.A polysiloxane Sm-Ch LC terpolymer with acrylate groups covalently linked to one of the mesogenic side-groups was successfully synthesized via a three-step route. The terpolymer obtained had an unexpectedly increased backbone chain length, and some formation of cyclic oligomer compounds was observed—both of which are side effects of the deprotection step. We propose the mechanism of these side reactions takes place through catalysis by PPTS. A temperature-responsive IR reflective coating was successfully fabricated from this terpolymer. This approach features easy processability using a bar-coating method. We have also found that a coating mediator can assist in the formation of planar alignment of cholesteric polysiloxane LCs during bar-coating. The fully-cured terpolymer coatings retained ~600 nm of reversible reflection band shift by heating and cooling. The partially retained temperature responsiveness of the coatings was still caused by the inherent Sm-Ch transitional effect of TP.The approach of using a polymer-stabilized polysiloxane Sm-Ch type LC polymer to fabricate a temperature-responsive photonic polymer is promising. By adjusting the chiral component faction in the Ch-LC siloxane, a reflection color in the visible region could also be achieved, further broadening the range of potential applications. However, as far as applications are concerned, the synthesis and the use of a nematic solvent provide limiting factors. Future optimizations of the material should also focus on fine-tuning the response temperature range and enhancing the mechanical robustness of the coating.


Rhizoma Coptidis is a popular phytomedicine for the treatment of type 2 diabetes in Asia, but its effective dose for diabetes treatment remains confused because of diverse origins. This study aimed to investigate the dose-response effects of Rhizoma Coptidis extract granules (RCEG), produced with standardized quality control, on hypoglycemic effects in patients with type 2 diabetes. We conducted a retrospective analysis of Chang Gung Research Database from January 01, 2008 to November 30, 2017. Outpatients visiting traditional Chinese medicine clinics and receiving RCEG for type 2 diabetes treatment were included. Plasma glucose, lipid, and other parameters were analyzed from 93 patients with a total of 737 visits within 60 weeks. Scatter plots with the LOESS analysis were used to explore the association between RCEG dose and hypoglycemic effect. The minimal effective dose was chosen to divide the study population into the high-dose and low-dose RCEG groups. Non-parametric tests were used for between-group and within-group comparisons. The multivariate nonlinear mixed-effects model was applied to access the effect of treatment length and groups simultaneously on the change of HbA1c and fasting plasma glucose. The “arule” package in R was used to present the network diagram of RCEG and other co-prescriptions. We first discovered a significant relationship between RCEG dose and HbA1c reduction when the dose reached 0.08 g/kg/day or higher. We thus defined 0.08 g/kg/day of RCEG as the minimum effective dose, and a threshold to separate patients into the high-dose (≥0.08 g/kg/d) and low-dose (<0.08 g/kg/d) RCEG groups. In the high-dose RCEG group, a significant decrease in total cholesterol and a trend toward triglyceride reduction were also noted. Patients more effectively responded to RCEG treatment if they had a higher initial HbA1c level, higher heart rates, better liver function tests, and better tolerance to the higher dose and treatment duration of RCEG. In addition, digestive/tonic/dampness draining formulas and blood regulation recipes were two of the most frequent co-prescriptions with RCEG. This study concluded that RCEG at a dose exceeding 0.08 g/kg/d had beneficial effects on glycemic and lipid control, without showing nephro- or hepatotoxicity, in patients with type 2 diabetes. Rhizoma Coptidis, commonly known as Huang-lian, was first recorded in Shennong’s Materia Medica in the eastern Han dynasty (25–220 AD), and it has been prescribed to treat diabetes-related symptoms “Xiaoke (消渴)” for the past 2000 years. According to traditional Chinese medical theory, Rhizoma Coptidis is cold in nature with bitter in taste, and has the function of purging fire toxins, clearing heat, and drying dampness. The phytochemical and therapeutic activities of Rhizoma Coptidis have been extensively reviewed in the literature  doctors prescribe Rhizoma Coptidis in the form of extract granules instead of herbal decoctions, because the former is covered by national health insurance. In addition, for Chinese herbal medicines (CHM), granule is the most prominent delivery format in the West. Rhizoma Coptidis extract granules (RCEG) produced by good manufacturing practice (GMP) manufacturers are available in many countries, including the United States, Canada, Switzerland, Malaysia, Singapore, and some European countries. Although Rhizoma Coptidis is widely prescribed for many diseases, including type 2 diabetes  is the largest multi-institutional, electronic medical record database in Taiwan . DiagnosThe Ethics Review Board of Chang Gung Medical Foundation in Taiwan approved this research (IRB No. 201701817B0). All data were collected from the CGRD and encrypted to protect the privacy of the patients. Medical records of outpatients who were diagnosed with type 2 diabetes and received the prescription of RCEG in the TCM clinics of Taipei, Linkou, and Taoyuan branches of Chang Gung Memorial Hospital from January 01, 2008 to November 30, 2017 were included. Diagnosis codes for type 2 diabetes include 250.00, 250.02, 250.40, 250.42, 250.50, 250.52, 250.60, 250.62, 250.80, 250.82, 250.90, and 250.92 in the International Classification of Disease, 9th Revision, Clinical Modification (ICD-9-CM) format, or E11.8, E11.9, E11.65, E11.69, E11.29, and E11.21 in the ICD-10-CM format. Patients were excluded from this study based on the following criteria: 1) no persistent treatment with RCEG for more than one month; 2) no HbA1c data or HbA1c <6.5% within three months before or two weeks after taking RCEG; 3) lack of HbA1c follow-up after taking RCEG for over one month; 4) uncertain, changed, or increased dosage of oral hypoglycemic agents (OHA) or insulin therapy during RCEG treatment; 5) failure to distinguish between high- and low-dose of RCEG due to no weight being recorded. The flowchart of the recruitment process is presented in Coptis chinensis Franch. The chemical composition of the RCEG was analyzed and profiled by using high performance liquid chromatography (HPLC) with photodiode array (PDA) detection  diagnosis codes; 2) date of visit; 3) name of CHM, dosage, frequency of administration, and treatment duration; 4) name and dose of OHA and insulin; 5) date and data of laboratory tests; 6) general information, including age, gender, and duration of diabetes; 7) body weight and height; 8) blood pressure and heart rate. There were very few patients receiving RCEG treatment for more than 60 weeks, so this study gated the maximum follow-up period to 60 weeks. Patients may have had different follow-up times because of the differing severity of their symptoms, drop outs, discontinuous RCEG treatment, or lack of laboratory data. The hypoglycemic response to RCEG treatment referred to changes of HbA1c level from pre-treatment to each patient’s last treatment. Scatter plots and the LOESS method were used to explore the association between RCEG dose and hypoglycemic effect in order to determine an optimal cutoff value for the dosage, thus allowing the study population to be divided into high-dose and low-dose RCEG groups for further analysis. The baseline characteristics of these two groups were analyzed. During RCEG treatment, the changes in HbA1c and fasting blood glucose at different time points were analyzed in both groups. The relative change was calculated by dividing the net change by the baseline value. Particularly in the high-dose group, the effect of treatment was measured based on the value changes from pre-treatment to the last available post-treatment due to limited data in some parameters. Co-prescription patterns and the daily dose of other CHM extract granules commonly combined with high-dose RCEG were also accessed. For each outpatient visit, the details of prescriptions, including extract granules of herbal formulas and single herbs, were recorded. In this study, a total of 636 prescriptions with high-dose RCEG were assessed. Frequent itemsets mining and rules of association were applied to evaluate the co-prescription patterns of CHM extract granules combined with RCEG. In the high-dose group, the relative change between the lowest HbA1c value and the first measured HbA1c value for each patient was calculated. The median relative change of HbA1c in the 12th week was –11.75%, and this value was set as a cutoff threshold to distinguish the high-response and low-response population within the high-dose RCEG group. The baseline characteristics, average daily dose and treatment duration of RCEG, and co-prescription patterns between the high- and low-response groups were analyzed.p < 0.05 was considered as the significance threshold for all two-tailed tests.Descriptive statistics were used to summarize the data. The relationship between RCEG dose and relative change of HbA1c was presented in the form of scatter plots with the LOESS analysis. Comparisons between groups were performed with the Mann-Whitney U-test for continuous parameters and Fisher's exact test for categorical parameters. The Wilcoxon signed rank test was used for pre- and post-comparison within the groups. The multivariate nonlinear mixed-effects model was applied to access the effect of treatment length and groups simultaneously on the change of HbA1c and fasting plasma glucose (FPG). The “arule” package in R was used to explore the common CHM combinations and present the CHM network in this study. A value of n = 77) and low-dose (<0.08 g/kg/d) RCEG population (n = 16). Baseline characteristics, including demographic records, anthropometric characteristics, vital signs, and laboratory data of the high-dose and low-dose RCEG groups are shown in There were 325 TCM outpatients diagnosed with type 2 diabetes according to the HbA1c ≥ 6.5% criteria and prescribed with RCEG from January 01, 2008 to November 30, 2017. Only 93 patients, who engaged in 737 visits within 60 weeks, were eligible and analyzed in this study . The anap < 0.001). There was a decrease in 2hPG from 247 to 162 mg/dl, which was not statistically significant, probably due to the limited sample size. Regarding the lipid profiles, a significant decline in total cholesterol from 180 to 154 mg/dl (p = 0.04) and a trend of reduction in triglycerides from 130 to 84 mg/dl (p = 0.07) were found after the high-dose RCEG treatment. However, there was no significant change in either high- or low-density lipoprotein cholesterol, possibly because of the sample size. In terms of safety parameters, including blood pressure, hepatic function, and renal function, high-dose RCEG exhibited a slightly beneficial effect on blood pressure control (p = 0.02) without nephro- or hepatotoxicity.The relative changes in HbA1c and FPG within the 60-weeks treatment period showed significant reductions in the high-dose RCEG group, but not in the low-dose group . In the It is possible that other CHMs, including single herbs, formulas, or a combination of both co-prescribed with RCEG, could influence glycemic control. Therefore, the daily dose of CHM and co-prescription pattern in the high-dose RCEG group were evaluated. The effects and indications of each co-prescribed CHM are summarized in  Hempen,. Unlike Rhizoma Coptidis  and low-dose (<0.08 g/kg/d) RCEG, manufactured by Sun Ten pharmaceutical company. This report illustrated that only RCEG in excess of 0.08 g/kg/d demonstrated therapeutic value in terms of anti-diabetic and anti-hypercholesterolemic effects for patients with type 2 diabetes.There are 128 chemical components in Coptidis , and theCoptidis . The ant in vivo . HoweverRhizoma Coptidis is prescribed for heat-clearing in the early and middle stage of T2DM  tract  tract . In geneCoptidis . AccordiCoptidis . The proCoptidis   can improve HbA1c, FPG and total cholesterol and is safe for patients with type 2 diabetes with or without OHA/insulin therapy. For each patient, the best outcome with minimum side-effects relies on tolerance to the dosage and duration of RCEG treatment. In addition, two of the most frequently prescribed CHMs alongside RCEG are digestive/tonic/dampness draining formulas and blood regulation recipes. This research serves as an evidence-based guideline for TCM doctors to treat type 2 diabetes with the proper dosage of RCEG and co-prescribed CHMs, and also provides alternative treatment options for patients to achieve better glycemic control by taking a phytomedicine. In the future, a large-scale randomized clinical trial with long-term follow-up should be launched to confirm these preliminary results.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The Ethics Review Board of Chang Gung Medical Foundation in Taiwan approved this research (IRB No. 201701817B0). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization, methodology and data curation, Y-HH and G-HL; formal analysis, T-YH; HPLC/PDA analysis of RCEG, M-CL; writing—original draft preparation, Y-HH; writing—review and editing, Y-HH and C-TH; validation, L-YY and Y-HW; supervision, Y-HW. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fig. 3d blank cell culture for MDA-MB-231 and MCF-7 appear identical to Fig. 4d blank cell culture for MDA-MB-231 and MCF-7 respectively.Fig. 3d charts appear to be identical to all Fig. 4d charts.Fig. 3f blank cell cultures appear to be the same as Fig. 4f blank cell cultures.Fig. 3g blank cell cultures appear to be the same as Fig. 4g blank cell cultures.Fig. 3i blank tissue samples appear to be the same as Fig. 4i blank tissue samples.The Editors-in-Chief have retracted this article . ConcernAdditionally, the Editors-in-Chief found that blank control values are missing in the raw data for Fig. 4a and that the variation between miRNA mimic and miRNA inhibitor appears to be unrealistically small. The Editors-in-Chief have therefore concluded that the data reported in this article are unreliable.The authors agree with the retraction but disagree with the wording of the retraction notice.